SUMMARYReactive oxygen species (ROS) are known to modulate activities of a host of kinases, phosphatases and transcription factors. Rutin and chlorogenic acid (CGA) are the major polyphenolic antioxidants present in the small molecular fraction of smokeless tobacco leaf extracts, as ascertained by reverse-phase high-pressure liquid chromatography (HPLC) and mass spectrometry. Levels of intracellular ROS in resting versus antigen±immunoglobulin E (IgE)-challenged murine mast cells were measured at 510 nm by¯uorescence-activated cell sorting (FACS) using carboxydichloro¯uorescein (DCFH-DA). Enhanced ROS production was observed in IgE-sensitized mast cells following antigenic challenge. Rutin and CGA reduced ROS levels in antigen±IgE-activated mast cells. Concomitantly, they also profoundly inhibited histamine release by these activated mast cells. In contrast, rutin and CGA augmented the inducible cytokine messages, i.e. interleukin (IL)-10, IL-13, interferon-c (IFN-c), IL-6 and tumour necrosis factor-a (TNF-a) in IgE-sensitized mast cells following antigen challenge. This study indicates that tobacco polyphenolic antioxidants that quench intracellular ROS, differentially affect two effector functions of antigen±IgE-activated mast cells. This model system may be employed to determine the molecular target of polyphenols. The potential role of these polyphenolic antioxidants on IgE-mediated allergy in vivo depends on a balance of their differential effects on mast cell activation.

Stlrnmal'yDendritic cells (DC), in general, and pulmonary DC, in particular, are a heterogeneous population of cells, their phenotype and function being dependent on their anatomic location, their state of activation, and the regulatory effect of locally secreted cytokines. Using a novel microdissection technique, the epithelium from the trachea and entire airway system was harvested, and the contained DC isolated at >90% purity. The phenotype and function of these airway DC (ADC) was compared to DC isolated, at >90% purity, from the parenchyma of the same lung. In contrast to lung DC (LDC), ADC did not express intercellular adhesion molecule 1 (ICAM-1) in situ, the amount of immune associated antigen (Ia) expressed was less (as determined by immunoperoxidase staining and immunopanning), and >50% of ADC displayed Fc receptors (FcR). The majority of LDC were ICAM-I+, <5% expressed FcK, and all were intensely Ia + . Airway DC were most numerous in tracheal epithelium, but they were also present in small numbers in the epithelium of the most distal airways. Their numbers increased in all segments of the tracheobronchial epithelium in response to the administration of IFN-% ADC were consistently more effective than LDC in presenting soluble (hen egg lysozyme) and particulate (heat-killed Listeria monocytogenes) antigens to antigen-sensitized T cells. By contrast, LDC were significantly more efficient in stimulating the proliferation of nonsensitized T cells in an autologous mixed leukocyte reaction. These data suggest that in normal animals, intraepithelial DC of airways share many attributes with Langerhans cells of the skin. Interstitial LDC, by contrast, reside in an environment where they may be exposed to a different set of regulatory factors and where they have progressed to a more advanced stage of differentiation than ADC. Both groups of DC are, however, heterogeneous, reflecting the continuous turnover that these cells undergo in the lung.

BackgroundAt present, it is highly controversial whether pure mast cells can serve as antigen presenting cells, and it is not known whether the capacity of antigen presenting function is temporally restricted to a particular subset of differentiated mast cells. Evidence is presented for a novel surface FcεRIhi , MHC II +, and c-kit + pure mast cell subset, temporally restricted as antigen-presenting cells in the immune axis of T-cell activation.ResultsBone marrow-derived mast cells (BMMC) cultured in the presence of IL-3 for three weeks are pure mast cells based on surface expression of lineage-specific marker, c-kit and FcεRI. Herein we present the first demonstration that approximately 98.7% c-kit + and FcεRI expressing BMMC, further depleted of any contaminated professional antigen-presenting cells, are still fully capable of presenting antigens, i.e., OVA protein, OVA peptide, and IgE-TNP-OVA, to OVA peptide-specific T-cell hybridomas. Notably, IgE-dependent antigen presentation is more efficient compared to that resulting from direct antigen uptake. Importantly, we present the novel finding that only surface FcεRIhi mast cells, also expressing surface MHC II exhibited antigen-presenting function. In contrast, surface FcεRIlo mast cells without expressing surface MHC II were not capable of antigen presentation. Interestingly, the antigen-presenting function of BMMC was irrevocably lost during the third and fourth week in IL-3 or SCF containing cultures.ConclusionsThis is the first observation to attribute a spatiotemporally restricted antigen-presenting function to a subset of three-week old pure BMMC expressing both high levels of surface FcεRI and surface MHC II. We propose that mast cells play an important role in immune deviating and/or sustaining the activation of infiltrating CD4 T-cells, and modulating T-cell mediated allergic inflammation via its flexibility to present antigens and antigen-IgE complexes.

In the adult mammalian lung, Ia+ dendritic cells (DC) constitute a significant population of immunologically potent accessory cells that are important in the regulation of immune responses to inhaled antigens. The newborn, in most species, displays an increased susceptibility to sensitization by inhaled antigens; whether an immaturity of pulmonary accessory cells is involved has not been determined. In the present study, the ontogeny and function of these cells were examined in fetal and newborn rats. Cells identified as DC in fetal and newborn rat lungs were Ia+, C11b+/-, OX41-, OX43-, W3/13-, W3/25-, and OX8-. They were characterized ultrastructurally by an eccentric, lobulated nucleus, a paucity of lysosomes, delicate cytoplasmic processes, and abundant membrane-associated Ia. Ia+ DC were first detected within the pulmonary mesenchyme at day 15 and by day 17 of gestation they were also present within the epithelium lining airways. The appearance of Ia+ DC preceded the migration of either T4 or T8 subclasses of T cells to the lung, the latter becoming significant only after birth, when the newborn was exposed to environmental antigens. In none of the fetal or newborn animals was Ia detected on alveolar type II cells. The accessory cell function of rat pulmonary DC, isolated from fetuses at 20 and 21 days of gestation and from newborns, was tested by an autologous mixed leukocyte reaction. At 20 and 21 days of gestation, pulmonary DC were 40 and 60% as effective, respectively, in stimulating cell proliferation in purified autologous adult splenic T cells as those isolated from adults.(ABSTRACT TRUNCATED AT 250 WORDS)

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Epidemiological data showed that total IgE and IL-4 levels in cigarette smokers were elevated, comparable to those in the asthmatics. The etiological agent(s) elevating IgE production are not clear. We evaluate whether tobacco polyphenols potentiate IgE production in a rodent model. Mice were fed with rutin or CGA in drinking water during antigen sensitization, followed by antigenic challenge i.p. in alum. CGA and rutin were also delivered in a bolus intraperitoneally or intranasally along with antigens during immunization. Antigen-specific IgE and IgG responses were measured. Enhancement of total IgE responses via i.p. and drinking routes can be achieved at concentrations as low as 0.1% CGA. Furthermore, IgG1 responses but not IgG2a and IgG2b were augmented, indicating a Th2 type of response by CGA. Moreover, both antigen-specific and serum IgE production can be achieved when CGA and antigenic challenges were delivered intranasally in the absence of alum. In contrast, nicotine does not enhance antigen-specific IgE production, and only marginally affects serum IgE levels. The more polarized Th2 development in CGA-treated mice may account for enhancement of both antigen-specific and total IgE responses. High levels of IL-4 but not IFN-gamma or IL-12, were observed in antigen-challenged mesenteric lymph nodes (MLN) cultures from CGA-treated mice. In contrast, significant levels of IL-4, IL-12, and IFN-gamma were observed in antigen-challenged cultures from nicotine-treated mice. This study shows that tobacco polyphenols, CGA and rutin potentiated IgE production in vivo. Polyphenolic antioxidants enhance Th2 development. We propose that IgE production and T cell dichotomy may be critically influenced by the redox microenvironment. Enhanced Th2 development and IgE production henceforth may counteract more severe Th1-mediated tissue damage triggered by environmental oxidative stress.

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Background The use of immune checkpoint inhibitors (ICI) has been associated with a 3-fold higher risk for cardiovascular events as compared to cancer patients who did not receive ICI. Therapies targeting vascular endothelial growth factor (VEGF) have also been associated with a wide range of cardiovascular events. The combination use of ICIs and VEGF inhibitors is currently approved as a treatment for patients with renal-cell carcinoma, hepatocellular carcinoma, non-small cell lung cancer, and endometrial cancer. Data are lacking whether the combination of ICIs and VEGF-targeted therapy is associated with an additional increase in cardiovascular events. Purpose To evaluate whether the combination use of ICI and VEGF targeted therapies are associated with a higher risk of cardiovascular events as compared to ICI therapy alone, we performed a retrospective matched case-control study. Methods Cases received both ICI and VEGF-targeted therapy (n=157), and control patients (n=157) only received ICI therapy. The primary outcome was a composite of cardiovascular events (myocardial infarction, coronary revascularization, ischemic stroke, deep venous thrombosis, and pulmonary embolism). Patients were censored at time of first event or at last date of follow up. Cox proportional hazard regression analysis was performed to calculate hazard ratio (HR) with 95% confidence interval (CI), counting only the first cardiovascular event. Results Baseline characteristics for the cases and controls are shown in Table 1. Overall cases (combination ICI and VEGF inhibitor) and controls (ICI alone) were not different with respect to age, type of cancer, and a prior history of any cardiovascular event. Cases received more ICI cycles as compared to controls (median of 7 [4–17] cycles vs. 4 [2–10] cycles, P&lt;0.001). Cases also had a longer follow-up time (334 [127–663] days vs. 201 [60–564] days, P=0.008) as compared to the control group. As compared to ICI alone, a similar risk for a composite cardiovascular event was observed in those who received both ICI and VEGF-targeted therapy (HR, 0.70 [95% CI, 0.39–1.25]; P=0.23, Table 1). In total, 21/157 patients had a composite cardiovascular event among the cases, who received the combination of ICI and VEGF inhibitor (9 DVT, one MI, 9 PE, two ischemic strokes) as compared to 25/157 among the controls, who received ICI alone (14 DVT, 3 MI, 7 PE, one ischemic stroke). The median time to event was not different between the two groups (126 [98–260] days vs. 145 [28–205] days, P=0.47). Conclusion We found that among 157 patients who received a combination of ICI and VEGF-targeted therapy and 157 matched control patients who only received ICI therapy, the risk for cardiovascular events was not different between the two groups. Funding Acknowledgement Type of funding sources: Public grant(s) – EU funding.